The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins, which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including eubacterial and eucaryotic hosts. The selection of an appropriate expression system often not only depends on the ability of the host cell to produce adequate yields of the protein in an active state, but also to a large extent may be governed by the intended end use of the protein.
One problem frequently encountered is the high level of proteolytic enzymes produced by a given host cell or in the culture medium. It has been suggested that one could provide host organism deprived of the ability of producing specific proteolytic compounds. For example, International Patent Application WO 90/00192 describes filamentous fungal hosts incapable of excreting enzymatically active aspartic proteinase, and EP 574 347 describes Aspergillus hosts defective in a serine protease of the subtilisin-type.
Metalloproteases have been isolated from a number of eucaryotic sources. Neutral metalloproteases, i.e. metalloproteases having optimal activity at neutral pH, isolated from strains of Aspergillus also have been reported. Neutral metalloproteases have been classified into two groups, NpI and NpII [Sekine; Agric. Biol. Chem. 1972 36 207-216]. Recently the nucleotide sequence of a neutral metalloprotease II cDNA from Aspergillus oryzae have been disclosed [Tatsumi H, Murakami S, Tsuji R F, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E and Motai H; Mol. Gen. Genet. 1991 228 97-103]. The nucleotide sequence of a neutral metalloprotease I cDNA from Aspergillus oryzae have never been disclosed.
Although metalloproteases have been reported, their role in relation to reducing the stability of the products obtained from these organisms have never been described.